Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of poly(A+) RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labelled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.
Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is posttranslationally modified by phosphorylation during viral infection. ICP27 has been shown to be phosphorylated on three serine residues, specifically serine residues 16 and 18, which are within casein kinase 2 (CK2) sites, and serine residue 114, which is within a protein kinase A (PKA) site. Phosphorylation is an important regulatory mechanism that is reversible and controls many signaling pathways, protein-protein interactions, and protein subcellular localization. To determine the role of phosphorylation in modulating the activities of ICP27, we constructed phosphorylation site mutations at each of the three serine residues. Single, double, and triple viral mutants were created in which alanine or glutamic acid was substituted for serines 16, 18, and 114. ICP27 phosphorylation site mutants were defective in viral replication and viral gene expression. Notably, ICP4-containing replication compartment formation was severely compromised, with the appearance of small ring-like structures that persisted even at late times after infection. Neither the colocalization of ICP27 with RNA polymerase II nor the formation of Hsc70 nuclear foci was observed during infection with the phosphorylation site mutants, both of which occur during wild-type HSV-1 infection. These data indicate that several key events in which ICP27 plays a role are curtailed during infection with ICP27 phosphorylation site mutants.Herpes simplex virus 1 (HSV-1) ICP27 is a multifunctional regulatory protein that interacts with a number of proteins and that binds RNA (32). The different activities of ICP27 are regulated in a temporal manner during infection. At early times, ICP27 is localized to the nucleus, whereas beginning about 5 to 6 h after infection, ICP27 continuously shuttles between the nucleus and cytoplasm. ICP27 mediates its nuclear activities through a series of protein-protein interactions. At very early times during infection, ICP27 recruits the predominantly cytoplasmic splicing factor kinase SRPK1 into the nucleus (34). This results in the aberrant phosphorylation of a family of essential splicing factors termed SR proteins, which are specifically phosphorylated by SRPK1. The inappropriate phosphorylation of SR proteins prevents their participation in spliceosome assembly, which causes splicing complex formation to stall, and host cell pre-mRNA splicing is impaired (15,34). This contributes to the shutoff of host protein synthesis (14). Also at early times after infection, ICP27 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNAP II) and causes RNAP II relocalization to sites of HSV-1 transcription/replication. This recruitment of RNAP II is necessary for highly active and efficient viral transcription (5). Beginning about 5 h after infection, ICP27 disassociates from splicing speckles, taking the cellular mRNA export factor Aly/REF with it to viral transcription/replication compartments (3, 4). ICP27 then binds viral tr...
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